Air shutoff agent for aqueous reagent and aqueous specimen, and method for storing aqueous reagent and aqueous specimen with stability

ABSTRACT

An air-barrier agent for an aqueous reagent or an aqueous specimen having as an effective component a mixture of a chain hydrocarbon and a silicone oil immiscible with the aqueous reagent as well methods of using and making the same.

TECHNICAL FIELD

The present invention relates to an air shutoff agent (air-barrieragent) used to stably store a reagent component or a substance to beanalyzed by preventing an aqueous reagent or an aqueous specimen frombeing in contact with air. More particularly, the present inventionrelates to an air-barrier agent which is used to stably store a reagentcomponent or a substance to be analyzed by layering the air-barrieragent over the surface of an aqueous reagent or aqueous specimen, andwhich does not soil a measuring section of an autoanalyzer duringrepeated use. The present invention also relates to a method for stablystoring an aqueous reagent or an aqueous specimen using the air-barrieragent.

BACKGROUND ART

At present, reagents used for clinical examinations are usually suppliedas a liquid or in freeze-dried form. Liquid reagents are used as is andfreeze-dried reagents are dissolved when used for measurement.Generally, these reagents are allowed to stand unsealed in a cool placeduring analysis using an autoanalyzer or during preservation.

Reagents in an unsealed state may exhibit insufficient stability.Specifically, measured values or sensitivity may decrease over timedepending on the properties of the reagent component. Moreover,coloration of the reagent or an increase in blank values frequentlyoccurs. Therefore, accuracy and precision which are the most importantproperties for diagnostic reagents may decrease over time. For example,a reagent using an OCPC method is used for determination of calcium inliving body fluid. Since monoethanolamine used as a base buffer solutionof the reagent absorbs CO₂ in air, the pH of the solution is shifted tothe acidic side, thereby affecting the measured values.

Depending on the type of specimen, a component to be analyzed may beeasily oxidized by oxygen in air. Such a specimen must be prevented frombeing in contact with air immediately after sampling in order to preventoxidization from occurring.

As technology for solving the above problems relating to reagents andspecimens, the present inventors have proposed a method of stabilizing areagent or the like by layering an air-barrier agent, which isimmiscible with the reagent and has a specific gravity lower than thatof the reagent over the reagent or the like (Japanese Patent ApplicationLaid-open No. 185927/1998).

This method can be applied to various reagents and specimens. However, aproblem may occur when this method is applied to a reagent used in anautoanalyzer. Specifically, the air-barrier agent layered over thereagent adheres to a reagent suction/regurgitate nozzle of theautoanalyzer. This may cause a slight amount of the air-barrier agent toflow into a reaction vessel together with the reagent when dischargingthe reagent into the reaction vessel. In the case of a recently usedreaction vessel made of a plastic, the air-barrier agent adheres to thewall of the reaction vessel, thereby soiling the surface of the wall byitself and by interaction with other substances.

Therefore, there has been a demand for an air-barrier substance whichdoes not soil a reaction vessel made of any material of an autoanalyzereven if the air-barrier agent flows into the reaction vessel whendischarging the reagent over which the air-barrier agent is layered.

DISCLOSURE OF THE INVENTION

The present inventors have conducted extensive studies to solve theseproblems. As a result, the present inventors have found that anair-barrier agent containing a chain (linear or branched) hydrocarbonand silicone oil does not soil a reaction vessel due to small affinitythereto, and can be widely used for a reaction vessel made of anymaterial. This finding has led to the completion of the presentinvention.

Specifically, the present invention provides an air-barrier agent for ananalytical reagent or specimen comprising a mixture of a chainhydrocarbon and silicone oil which is immiscible with an aqueous reagentor an aqueous specimen and has a specific gravity lower than that of thereagent or specimen.

The present invention also provides a method for stably storing anaqueous analytical reagent or an aqueous specimen comprising layeringthe above mixture over the surface of the aqueous analytical reagent oraqueous specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparative view showing soiling conditions in the case ofusing a present invention product 1 and a reference product 1.

FIG. 2 is a view comparatively showing coloration over time of a reagent1 of a triglyceride measuring reagent containing a color developer towhich an air-barrier agent of the present invention is added and thereagent 1 containing no air-barrier agent.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, a mixture of a chain hydrocarbon and siliconeoil is used as the air-barrier agent. This mixture must be immisciblewith an objective aqueous reagent or aqueous specimen (hereinaftercalled “reagent or specimen”) and have a specific gravity (density)lower than that of the reagent or the like. Generally, the specificgravity of a reagent such as a reagent used for clinical examination isabout 1. Therefore, the specific gravity of the mixture is preferablyabout 0.7-0.95, and particularly preferably about 0.86 or less. It ispreferable that the mixture be a liquid at room temperature and have aspecific gravity differing from that of the reagent in the range fromabout 0.05 to 0.3.

As the chain hydrocarbon among the components of the mixture, a chainhydrocarbon with a viscosity of less than 37 cSt and a flash point of46° C. or more is used. Specific examples include octane, nonane,decane, undecane, dodecane, tridecane, tetradecane, and the like. Ofthese, a chain hydrocarbon with a flash point of 80° C. or more ispreferable in view of ease in handling in practical application.

As the silicone oil which is the other component of the mixture, apolyalkylsiloxane, polyalkylphenylsiloxane, polyalkyl hydrogen siloxane,and the like can be used. It is preferable to use a lineardimethylpolysiloxane which is commercially available as silicone oil,which is a mixture of linear dimethylpolysiloxanes having differentmolecular weights and shown by the following formula in which n isvarious values.

As the linear dimethylpolysiloxane, linear dimethylpolysiloxanes havingvarious kinematic viscosities of 100 cSt or less are known. Thekinematic viscosity of the linear dimethylpolysiloxane is preferablyabout 1.5-100 cSt, and still more preferably 3-50 cSt.

The mixture may be prepared using the chain hydrocarbon and the siliconeoil so that the silicone oil content in the mixture is 1-99%, preferablyabout 10-90%, and still more preferably 30-50%.

When using the mixture of the chain hydrocarbon and the silicone oilthus prepared as the air-barrier agent, the mixture may be layered overthe surface of an aqueous reagent or an aqueous specimen which is in theform of a liquid or a solution obtained by dissolving a freeze-driedproduct. There are no specific limitations to the amount of theair-barrier agent to be used. It is preferable to use the air-barrieragent in an amount from 0.03 ml to 1.0 ml per 1 cm² of the surface ofthe reagent (surface in contact with air with no air-barrier agent layerformed).

A stable reagent unit may be prepared by filling a container with aliquid reagent using a conventional method and forming a layer of themixture of the chain hydrocarbon and the silicone oil on the uppersurface of the reagent. Alternatively, a stable reagent unit may beprepared by adding the mixture of the chain hydrocarbon and the siliconeoil to a container and forming a layer of the mixture by injecting aliquid reagent into the container. As a material for the container forthe reagent unit, glass, metals, plastics, and the like can be used. Itis preferable to use a container which does not allow air to permeateand has a narrow opening.

In the present invention, it is important to use the reagent for testsor examinations by removing only the reagent or the like from thecontainer using a reagent suction nozzle of an autoanalyzer, pipette,needle, or the like without mixing the reagent or specimen with theair-barrier agent.

If surfactants or other materials which may emulsify the air-barrieragent are present in the reagent, the effect of the present inventionmay become insufficient. If any components of the reagent having somefunction in the measurement have extremely high affinity to theair-barrier agent, such components if moved to the air-barrier agentlayer may impair the effects of the reagent. Care should be taken toprevent such occurrence.

INDUSTRIAL APPLICABILITY

The air-barrier agent of the present invention prevents the reagent orthe like from being in contact with air by layering the air-barrieragent over the surface of the reagent or the like. Therefore, theair-barrier agent can provide the reagent or the like with excellentstorage stability, thereby enabling measurement for a long period oftime.

The air-barrier agent of the present invention does not soil materialswhich may be used for a reaction vessel of an autoanalyzer, such asglass, quartz, plastics (polypropylene, polystyrene, methacrylate,polymethylpentene, and the like) Therefore, the air-barrier agent can berepeatedly used in the autoanalyzer.

As described above, the present invention can effectively prevent thereagent or the like from being in contact with air and does not soil thereaction vessel of the autoanalyzer. Therefore, the present inventioncan be advantageously applied to a reagent used for analysis using theautoanalyzer, such as an alkaline phophatase measuring reagent, calciummeasuring reagent, bile acid measuring reagent, creatinine measuringreagent, and triglyceride measuring reagent or specimen.

EXAMPLES

The present invention will be described in detail by examples, whichshould not be construed as limiting the present invention.

Example 1 Application to Calcium Measuring Reagent

Isoparaffin and silicone (polydimethylsiloxane, 200R FLUID, kinematicviscosity: 5 cSt, manufactured by Aldrich) were mixed at a ratio of 6 to4 to prepare an air-barrier agent (present invention product 1).

This air-barrier agent was layered over a reagent 1 of a calciummeasuring reagent according to a composition given below. Using aHitachi 7170 Automatic Analyzer, 150 μl of the reagent 1 was added to 4μl of a specimen, and the mixture was stirred and then incubated at 37°C. for five minutes. After the addition of a reagent 2, the mixture wasincubated at 37° C. for five minutes. The absorbance of the mixture at600 nm was then measured.

The measurement was repeated four times continuously using the samereaction vessel (made of polymethylpentene plastic). After themeasurements, the degree of soiling of the reaction vessel was observedwith the naked eye and evaluated according to standards given below.FIG. 1 shows the soiling conditions.

As a reference, a reference product 1 prepared using a reagent 1 of thepresent invention product containing only isoparaffin in instead of the6:4 mixture of isoparaffin and silicone was used.

(Reagent Composition of Present Invention Product 1)

Reagent 1

Monoethanolamine: 0.5 M

Mixture of isoparaffin and silicone (ratio=6:4): 3 ml

(Per 70 ml bottle)

Reagent 2

o-Cresolphthalein complexon: 0.13 mM

8-Hydroxy-5-sulfonic acid: 10 mM

Acetic acid buffer solution: 0.24 M (pH 6.0)

(Reagent 1 of reference product 1)

Monoethanolamine: 0.5 M

Isoparaffin: 3 ml

(Per 70 ml bottle)

(Evaluation Standards for Soiling of Reaction Vessel)

Evaluation Condition − No soiling ± Very slight soiling + Slight soiling++ Soiled +++ Soiled significantly

(Results)

The evaluation results of the soiling of the reaction vessel are shownin Table 1.

TABLE 1 Reagent Degree of soiling Present invention product 1 −Reference product 1 +++

As is clear from these results, the reaction vessel exhibited no soilingafter four continuous measurements using the present invention product1. On the contrary, the inner wall of the reaction vessel was soiledwhen using the reference product 1.

As shown in FIG. 1, no soiling was recognized in the reaction vesselafter measurements using the present invention product 1 (right in FIG.1). On the contrary, soiling was recognized at the center of thereaction vessel when using the reference product (left in FIG. 1).

As described above, the air-barrier agent of the present invention whichuses the mixture of a chain hydrocarbon and silicone does not cause thereaction vessel to be soiled during repeated use.

Example 2 Application to Alkaline Phosphatase Measuring Reagent

Each mixture of a chain hydrocarbon and silicone oil (air-barrier agent)was layered over a reagent 1 of an alkaline phosphatase measuringreagent having a composition given below. The mixture was allowed tostand unsealed in a cool place (8° C.). Alkaline phosphatase activity inhuman blood serum was sequentially measured using this alkalinephosphatase measuring reagent according to a method given below. Changesin the measured values were compared and examined. The results are shownin Table 2.

(Reagent Composition)

Reagent 1

Ethylaminoethanol buffer solution: 1.0 M (pH 9.9)

Magnesium chloride: 0.5 mM

Air-barrier agent* (see Table 2): 0.2 ml/cm²

Reagent 2

p-Nitrophenylphosphosphate: 15 mM

* Amount added per 1 cm2 of open area of reagent container (hereinafterthe same)

(Measuring Method)

260 μl of the reagent 1 was added to 4 μl of a specimen. The mixture wasstirred and then incubated at 37° C. for five minutes. After theaddition of 130 μl of the reagent 2, the mixture was stirred. Using areagent blank as a reference, changes in absorbance of the mixture at awavelength of 450 nm were measured at 37° C. from two to five minutesusing a Hitachi 7150 Automatic Analyzer. A physiological saline solutionwas used as the reagent blank. The alkaline phosphatase (ALP) activitywas calculated from a conversion factor derived from a molecularextinction coefficient of p-nitrophenylphosphate, which was the reactionproduct, and the variation in absorbance.

(Results)

TABLE 2 ALP measured value (U/l) Air-barrier agent 0 day 7 days 14 days22 days Undecane + silicone 444 442 443 442 Dodecane + silicone 442 444441 443 Tridecane + silicone 442 441 443 442 Tetradecane + silicone 443441 442 444 Isoparaffin + silicone 443 444 442 442 No addition 443 385329 237 (reference)

As is clear from these results, the measured values significantlydecreased over time in the case where the air-barrier agent was notadded (reference). On the contrary, the measured values showed nodecrease after 22 days in the case where the air-barrier agent of thepresent invention was used. Specifically, the alkaline phosphataseactivity can be precisely measured for a long period of time by usingthe air-barrier agent of the present invention.

Example 3 Application to Bile Acid Measuring Reagent

A mixture of undecane and silicone (ratio=6:4) was added to a reagent 2of a bile acid measuring reagent as an air-barrier agent according to acomposition given below (present invention product 2). The mixture wasallowed to stand unsealed in a cool place. A bile acid standardizedsolution (50 μM) was sequentially measured using the reagent 2 of thebile acid measuring reagent. Changes in the measurement sensitivity wascompared and examined. The results are shown in Table 3. As a reference,a reagent 2 excluding the above undecane/silicone mixture (referenceproduct 2) was used.

(Reagent Composition)

Reagent 1

Oxidized β-thionicotinamide adenine dinucleotide: 0.99 mg/ml

Glycine buffer solution: 0.03 M (pH 4.0)

Reagent 2

Reduced β-nicotinamide adenine dinucleotide: 5.04 mg/ml

3α-Hydroxysteroid dehydrogenase: 9.5 U/ml

Mixture of undecane and silicone: 0.3 ml/cm²

Diethanolamine buffer solution: 0.2 M (pH 9.2)

(Measuring Method)

260 μl of the reagent 1 was added to 3 μl of a specimen. The mixture wasincubated at 37° C. for five minutes. After the addition of 130 μl ofthe reagent 2, the mixture was stirred. Using a reagent blank as areference, changes in absorbance of the mixture at a wavelength of 405nm was measured at 37° C. from one to three minutes using a Hitachi 7150Automatic Analyzer. A physiological saline solution was used as thereagent blank.

(Results)

TABLE 3 Bile acid measurement sensitivity (mAbs/min) Present inventionReference Preservation product 2 product 2  0 day 34 34  8 days 33 24 16days 33 22 24 days 32 19

As is clear from these results, the measurement sensitivitysignificantly decreased over time in the case where the air-barrieragent was not added (reference product 2). On the contrary, themeasurement sensitivity showed no decrease in the case where theair-barrier agent of the present invention was used (present inventionproduct 2). Specifically, bile acid can be precisely measured for a longperiod of time by the method using the air-barrier agent of the presentinvention.

Example 4 Application to Creatinine Measuring Reagent

A mixture of tridecane and silicone (ratio=6:4) was added to a reagent 1of a creatinine measuring reagent as an air-barrier agent (presentinvention product 3) according to a composition given below. The mixturewas allowed to stand unsealed in a cool place. A creatinine standardizedsolution (10.0 mg/dl) was sequentially measured using the reagent 1 ofthe creatinine measuring reagent. Variation in the measurementsensitivity was compared and examined. The results are shown in Table 4.As a reference, a reagent 1 excluding the above tridecane/siliconemixture (reference product 3) was used.

(Reagent Composition)

Reagent 1

Sodium hydroxide: 0.2 M

Mixture of tridecane and silicone: 0.2 ml/cm²

Reagent 2

2,4,6-Trinitrophenol: 28.4 mM

(Measuring Method)

300 μl of the reagent 1 was added to 15 μl of a specimen. The mixturewas stirred and then incubated at 37° C. for five minutes. After theaddition of 75 μl of the reagent 2, the mixture was stirred and thenincubated at 37° C. for five minutes. Using a reagent blank as areference, absorbance at a wavelength of 505 nm was measured using aHitachi 7150 Automatic Analyzer. A physiological saline solution wasused as the reagent blank.

(Results)

TABLE 4 Creatinine measurement sensitivity (mAbs/100 mg/dl) Presentinvention Reference Preservation product 3 product 3  0 day 931 930  8days 930 371 16 days 931  94

As is clear from these results, the measurement sensitivitysignificantly decreased over time in the case where the air-barrieragent was not added (reference product 3). On the contrary, themeasurement sensitivity showed no decrease in the case where theair-barrier agent of the present invention was used (present inventionproduct 3). Specifically, creatinine can be precisely measured for along period of time by the method using the air-barrier agent of thepresent invention.

Example 5 Application to Triglyceride Measuring Reagent

An air-barrier agent (mixture of tetradecane and silicone (ratio=6:4))was added to a reagent 1 containing a color developer of a triglyceridemeasuring reagent (present invention product 4) according to acomposition given below. This product was allowed to stand unsealed in acool place. The degree of coloration over time of the reagent 1 wascompared with a reagent 1 excluding the above tetradecane/siliconemixture (reference product 4) using a measuring method given below. Theresults are shown in FIG. 2.

(Reagent Composition)

Reagent 1

Glycerol kinase: 900 U/l

Glycerol 3-phosphate oxidase: 3800 U/l

Peroxidase: 1950 U/l

Sodium 3,5-dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl) aniline: 1.8 mM

Mixture of tetradecane and silicone: 0.2 ml/cm²

Good buffer solution: 0.2 M (pH 6.5)

(Measuring Method)

10 ml of the reagent 1 was allowed to stand unsealed in a cool place for40 days. Using a reagent blank as a reference, an absorption spectrum ofthe reagent at a wavelength from 400 nm to 800 nm was measured using aBeckman DU640 spectrophotometer. As the reagent blank, distilled waterwas used.

(Results)

As is clear from FIG. 2, in the case where the air-barrier agent was notadded (reference product 4), oxygen in air was absorbed into thereagent, thereby causing oxidization of the color developer to proceed.As shown in FIG. 2(a), significant blue coloration exhibiting anabsorption peak at 590 nm was observed.

On the contrary, in the case where the air-barrier agent was used(present invention product 4), no oxidization and coloration of thereagent were observed, as shown in FIG. 2 (b) Specifically, according tothe method using the air-barrier agent of the present invention,coloration of the triglyceride measuring reagent can be prevented,whereby the reagent can be stably stored for a long period of time.

What is claimed is:
 1. An air-barrier agent comprising a mixture of achain hydrocarbon and silicone oil, wherein the silicone oil has akinematic viscosity of 100 cSt or less, and the mixture is immisciblewith an aqueous reagent or an aqueous specimen, and has a specificgravity lower than that of the aqueous reagent or the aqueous specimen.2. The air-barrier agent according to claim 1, wherein the chainhydrocarbon comprises at least one member selected from the groupconsisting of a liquid paraffin, isoparaffin, decane, undecane,dodecane, and tridecane.
 3. The air-barrier agent according to claim 1,wherein the silicone oil comprises at least one member selected from thegroup consisting of polyalkylsiloxane, polyalkylphenylsiloxane, andpolyalkyl hydrogen siloxane.
 4. The air-barrier agent according to claim3, wherein the content of the silicone oil is from 1 to 99% based on themixture of the chain hydrocarbon and the silicone oil.
 5. Theair-barrier agent according to claim 1, wherein the mixture of the chainhydrocarbon and the silicone oil has a specific gravity of from 0.7 to0.95.
 6. The air-barrier agent according to claim 5, wherein the contentof the silicone oil is from 1 to 99% based on the mixture of the chainhydrocarbon and the silicone oil.
 7. The air-barrier agent according toclaim 1, wherein the content of the silicone oil is from 1 to 99% basedon the mixture of the chain hydrocarbon and the silicone oil.
 8. Theair-barrier agent according to claim 1, wherein the aqueous reagentcomprises at least one, member selected from the group consisting ofalkaline phosphatase measuring reagent, calcium measuring reagent, bileacid measuring reagent, creatinine measuring reagent and triglyceridemeasuring reagent.
 9. The air-barrier agent according to claim 1,wherein the chain hydrocarbon and the silicone oil is present at a ratioof from 6 parts of the chain hydrocarbon to 4 parts of the silicone oil.10. A method for storing an aqueous reagent or an aqueous specimen,comprising: contacting the air-barrier agent of claim 1 with the aqueousreagent or the aqueous specimen.
 11. The method of claim 10, wherein theaqueous reagent comprises at least one member selected from the groupconsisting of alkaline phosphatase measuring reagent, calcium measuringreagent, bile acid measuring reagent, creatinine measuring reagent andtriglyceride measuring reagent.
 12. A composition comprising theair-barrier agent of claim 1; and an aqueous reagent or aqueousspecimen.
 13. The composition according to claim 12, wherein theair-barrier agent is present in an amount ranging from 0.03 to 1.0 mlper 1 cm² of surface area of the aqueous reagent or the aqueousspecimen.
 14. An autoanalyzer comprising the composition of claim 12,and a reaction vessel.
 15. The autoanalyzer of claim 14, wherein thereaction vessel comprises at least one material selected from the groupconsisting of glass, quartz and plastic.
 16. An autoanalyzer comprisingthe air-barrier agent of claim 1 and a vessel.
 17. A method for storingan aqueous reagent, or an aqueous specimen comprising: contacting amixture of a chain hydrocarbon and a silicone oil with the aqueousreagent or the aqueous specimen, wherein the silicone oil has akinematic viscosity of 100 cSt or less, and the mixture is immisciblewith the aqueous reagent or the aqueous specimen, and has a specificgravity lower than the aqueous reagent or aqueous specimen.